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Intermediate reactions in the binding of aminoacyl-transfer ribonucleic acid to rat liver ribosomes. Formation and properties of an aminoacyl-transfer ribonucleic acid-transferase I complex.

ABSTRACT: 1. Transferase I of rat liver binds aminoacyl-tRNA to form a relatively stable complex, which is retained on cellulose nitrate filters. This reaction proceeds at both 0 degrees C and 37 degrees C and is inhibited by GTP. The resulting product is stabilized by GTP and Mg(2+). 2. Only very low quantities of deacylated tRNA are bound by transferase I. 3. Methods are described for the preparative isolation of the transferase I-aminoacyl-tRNA complex from incubation mixtures by using ion-exchange procedures. 4. The transferase I-aminoacyl-tRNA complex becomes readily bound to ribosomes. The presence of Mg(2+) is essential for the binding. GTP stimulates this reaction but is not absolutely required. 5. It is concluded that the formation of the transferase I-aminoacyl-tRNA complex may be the primary reaction in the binding of aminoacyl-tRNA to mammalian ribosomes and that, unlike in bacterial systems, GTP is not absolutely required for this step.

SUBMITTER: Hradec J

PROVIDER: S-EPMC1178500 | biostudies-other | 1972 Feb

REPOSITORIES: biostudies-other

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Intermediate reactions in the binding of aminoacyl-transfer ribonucleic acid to rat liver ribosomes. The role of guanosine triphosphate.

Project description:1. Transferase I from rat liver binds relatively low quantities of GTP when incubated with this nucleotide in the absence of aminoacyl-tRNA. 2. Transferase I reacts with both aminoacyl-tRNA and GTP to form a relatively stable complex that is retained on cellulose nitrate filters. The ternary complex transferase I-aminoacyl-tRNA-GTP is also formed when the transferase I-aminoacyl-tRNA complex is incubated with GTP or during the incubation of the transferase I-GTP complex with aminoacyl-tRNA. Synthesis of this complex does not require the presence of Mg(2+). 3. In the presence of Mg(2+) the ternary complex becomes readily bound to ribosomes without requirements for any other cofactors. 4. An extensive cleavage of GTP takes place when aminoacyl-tRNA becomes bound to ribosomes. 5. The low interdependence of reactions leading to the formation of transferase I complexes with aminoacyl-tRNA and GTP indicates that the mechanisms of the binding reaction in mammalian systems may be different from those in bacterial cells.

| S-EPMC1178501 | biostudies-other

Intermediate reactions in the binding of aminoacyl-transfer ribonucleic acid to rat liver ribosomes. the interaction of cholesteryl 14-methylhexadecanoate.

Project description:1. Transferase I from rat liver extracted with iso-octane binds significantly less aminoacyl-tRNA than the non-extracted enzyme. The original activity can be fully restored by the addition of cholesteryl 14-methylhexadecanoate. The binding capacity for GTP is not affected by the extraction. 2. In the presence of extracted transferase I the binding of aminoacyl-tRNA to ribosomes is decreased to 11-26% and the simultaneous binding of GTP to 32-43%. Cholesteryl 14-methylhexadecanoate induces a full reactivation of the extracted enzyme in both respects. 3. Extracted complexes A (aminoacyl-tRNA-GTP-transferase I) become bound to ribosomes to the same extent as the corresponding non-extracted preparations. 4. It is concluded that cholesteryl 14-methylhexadecanoate interacts with the binding site of transferase I for aminoacyl-tRNA and secondarily with that for GTP. It does not affect the binding site for ribosomes.

| S-EPMC1178546 | biostudies-other

Myocardial aminoacyl-transfer-ribonucleic acid synthetase and aminoacyl-transferring enzyme activity.

Project description:The properties of cytoplasmic aminoacyl-tRNA synthetase and aminoacyl-transferring enzymes in the myocardium were examined and methods for the assay of the activity of these enzyme systems were developed. Aminoacyl-tRNA synthetase activity was measured from the rate of incorporation of (14)C-labelled amino acid into aminoacyl-tRNA. Transferase activity was measured from the rate of incorporation of amino[(14)C]acyl-tRNA into protein in the presence of a standard preparation of hepatic ribosomes. Aminoacyl-tRNA synthetase activity is labile once the heart has been homogenized, whereas transferase activity is stable. The source of energy for synthetase activity is ATP; that for transferase is GTP. Transferase activity was inhibited by puromycin and stimulated by dithiothreitol, whereas synthetase activity was unaffected.

| S-EPMC1178388 | biostudies-other

Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Cognate transfer ribonucleic acid as a ligand.

Project description:The use of tRNA affinity columns for the purification of aminoacyl-tRNA synthetases was investigated. A purification method for valyl-tRNA synthetase from Bacillus stearothermophilus is described that uses two affinity columns, one containing the pure cognate tRNA, and the other containing all tRNA species except the cognate tRNA. A method for the rapid preparation of the two columns was developed, which does not require prior isolation of cognate tRNA but makes use of the ability of the target synthetase to select its cognate tRNA. The usefulness of tRNA columns is compared with that of affinity columns derived from the aminoalkyladenylate reported in the preceding paper [Clarke & Knowles (1977) Biochem J. 167, 405-417].

| S-EPMC1183673 | biostudies-other

Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Small organic ligands.

Project description:The usefulness of affinity chromatography for the purification of aminoacyl-tRNA synthetases was explored by using column ligands derived from the corresponding amino acid and aminoalkyladenylate, a non-labile analogue of the aminoacyladenylate reaction intermediate. Four modes of attachment of the aminoalkyladenylate to Sepharose were studied. The interaction between amino acid derivatives and the corresponding aminoacyl-tRNA synthetases is too weak to allow their use as ligands for affinity chromatography. Attachment of the aminoalkyladenylate via the alpha-nitrogen atom of the amino acid or via C-8 of the nucleotide abolishes synthetase binding, and immobilization via the oxidized ribose ring is only marginally useful. However, attachment of the aminoalkyladenylate to the matrix via N-6 of the nucleotide allows strong and specific synthetase binding, and the use of such columns permits the isolation of homogeneous synthetase from crude mixtures. The effect of non-specific adsorption and the utility of pre-columns and of specific substrate elution are investigated and discussed.

| S-EPMC1183672 | biostudies-other

Binding of chloramphenicol and a fragment of aminoacyl-transfer ribonucleic acid to ribosomes and a ribosome precursor from a mutant of Escherichia coli.

Project description:During exponential growth, the mutatn strain Escherichia coli 15-28 accumulates 47S particles, which are unusual precursors to 50S ribosomal subunits. The 47S particles have little ability to bind chloramphenicol, but binding of a fragment of aminoacyl-tRNA is about half that by completed subunits. The 70S (and 50S) ribosomes of strain 15-28 and its parent (strain 15TP) do not differ in chloramphenicol binding. Although ribosomes from the mutant are less able than those from the parent to bind the fragment, this difference is not as marked as was found previously [Sims & Wild (1976) Biochem. J. 160, 721-726] for the binding of an analogue of peptidyl-tRNA and for peptidyltransferase activity. The altered activities may arise because strain 15-28 misassembles 50S subunits of altered conformation and because the few proteins that 47S patricles lack have vital functions in some of the partial reactions of protein synthesis.

| S-EPMC1185724 | biostudies-other

The extraction and assay of aminoacyl-transfer-ribonucleic acid synthetases of tobacco leaf.

Project description:1. Aminoacyl-transfer-RNA synthetase activity in extracts prepared from tobacco leaf was increased 3-5-fold when sodium thioglycollate (30mm) and magnesium chloride (16mm) were included in the extraction medium. Omitting sucrose (0.45m) from the extraction medium did not alter the activity. 2. Activity was a linear function of enzyme concentration up to 1 disk (30mg. fresh wt.)/ml. and was not affected by dialysis at any concentration. 3. Activity increased about 13-fold above control values when a mixture of 21 amino acids and amides (1mm) was added to the reaction mixture. 4. Under the conditions used in the standard assay for aminoacyl-transfer-RNA synthetase activity K(m) (ATP) was 0.65mm and K(m) (l-amino acids) was 70mum. 5. Activity above the control value was found with all amino acids and amides tested except alanine, arginine, glutamic acid, glutamine and hydroxyproline. Activity was highest with leucine, isoleucine, valine, cysteine and histidine. Total activity with a mixture of 21 amino acids and amides was 20% lower than the total activity of the enzymes assayed separately.

| S-EPMC1270057 | biostudies-other

Preparation of polyribosome aminoacyl-transfer ribonucleic acid from the muscle of chick embryos.

Project description:A procedure for preparing polyribosome aminoacyl-tRNA free from contamination by supernatant aminoacyl-tRNA and free amino acids is described. Important features of the procedure are the use of acidic buffers to help protect the amino acid-tRNA linkage and the inclusion of sodium dodecyl sulphate, to inhibit ribonuclease activity. The specific radioactivity of polyribosome aminoacyl-tRNA is high within 30s and reaches a maximum in 2 1/2 min, well ahead of polyribosome peptides which, as described by Herrmann et al. (1971), attain maximum specific radioactivity in about 10 min.

| S-EPMC1165474 | biostudies-other

Factors controlling aminoacyl-transfer-ribonucleic acid synthesis in vitro by a plant system.

Project description:1. Factors affecting aminoacyl-tRNA synthesis in vitro by cell-free preparations from bean leaves were investigated. 2. Evidence was obtained that optimum concentrations as well as correct ratios of Mg(2+) and ATP are required for aminoacyl-tRNA synthesis in the bean-leaf system. 3. The results indicated that pH is a controlling factor having differential effects on the formation of individual aminoacyl-tRNA species. The possible micro-regulatory function of pH in protein synthesis in vivo is discussed with special reference to alanyl-tRNA formation. 4. Very low rates of alanine-stimulated pyrophosphate exchange were observed in the absence of tRNA. This observation is discussed relative to proposals about the mechanism of aminoacyl-tRNA synthesis.

| S-EPMC1176597 | biostudies-other

Rates of aminoacyl-transfer-ribonucleic acid synthesis in vivo and in vitro by bean leaves.

Project description:1. A procedure for measuring rates of aminoacyl-tRNA synthesis in vitro and in intact leaves is presented. 2. Leaf discs showed rates close to those of intact leaves. 3. Cell-free preparations showed similar rates when assayed by pyrophosphate exchange, but actual aminoacyl-tRNA formation rates appeared to be much lower. Evidence is presented that dilution of supplied labelled amino acids was a major factor causing the low apparent rates. 4. Attempts to strip endogenous amino acids from plant tRNA resulted in low acceptor capability of the tRNA.

| S-EPMC1179044 | biostudies-other

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